Study On The Biosynthesis Of Pimelic Acid

Pimelic acid is an important dicarboxylic acid,which is broadly applied in various fields,including chemical industry,medicine,coordination chemistry and other fields.Also,it is an important raw material for the preparation of biotin,lubricating oil and polyamide.At present,the industrial production of pimelic acid is mainly through the cleavage of salicylic acid,which has the disadvantages of strict reaction conditions,complex technological process and serious environmental pollution.Recently,the biological synthesis of pimelic acid has received widespread attention,and the biosynthesis mechanism of pimelic acid has been gradually clarified.However,the yield of pimelic acid in engineering strains was still low in the existing reports,which is not conducive to industrial production.In order to solve this problem,this study has done the following:（1）Constructing the biosynthesis pathway of pimelic acid.Firstly,based on the previous study,E.coli B and B3 were used to verify the feasibility of reverse adipate-degradation pathway（RADP）for the biosynthesis of pimelic acid.In order to inhibit the downstream metabolic pathway of pimelic acid,CRISPR-Cas9 was used to knock out bioF of strain B and B3,generating host strains B1 and B4.Then five enzymes of the RADP were overexpressed in four host strains.The results of shake flask fermentation showed that deleting bioF of E.coli individually promoted the synthesis of pimelic acid and B1-146 had the highest pimelic acid titer at 12.0 mg·L^-1 when 50 mM glutaric acid was added.（2）Enhancing the supply of glutaryl-CoA.In this study,glutaric acid transporter PA5530 and coenzyme A transferase Cat1 and CaiB were expressed in strain B1-146 for the purpose of increasing the level of intracellular glutaryl-CoA.The results of shake flask fermentation showed that the glutaric acid transporter PA5530 had a positive effect on the synthesis of pimelic acid.Through the optimization of the concentration of glutaric acid added,it was concluded that when the concentration of glutaric acid added was 10 mM,the titer of pimelic acid was the highest in the strain B1-146P who expressed PA5530.（3）Optimizing the synthetic pathway of pimelic acid by the combination of isoenzymes.Firstly,the isoenzymes of RADP pathway were excavated through literature research,and the BIOZ pathway was constructed to verify the feasibility for the synthesis of pimelic acid via expressing these enzymes in E.coli B1.Then,these isoenzymes of the RADP and BIOZ pathway were combined.Through shake flask fermentation,it was found that when bioZ,fab G,Tfu＿0067,Tfu＿1647 and Tfu＿2576-2577were expressed,the yield of pimelic acid of strain B1 was the highest(36.7 mg·L^-1),which was also the highest yield of pimelic acid synthesized by Claisen condensation reaction.（4）Screening and fermentation optimization of pimelic acid producing strain.In this study,a pimelic acid-producing Enterobacter hormaechei Bpa-3 was obtained by screening in 48-well plate and shake flasks,of which the pimelic acid production was 115.4 mg·L^-1 during shake flask fermentation.Subsequently,the composition of culture medium,the amount of inoculum and the concentration of inorganic salt added were optimized respectively.As a result,when glycerol was used as carbon source,tryptone as nitrogen source,inoculum amount was 4%and 30 mM KH₂PO₄was added,the titer of pimelic acid was 217.3 mg·L^-1,which was 1.89-fold than that before optimization.